1. Purebred American Angus calves were used to evaluate the extent to which variation in the TLR4 locus was associated with pink eye. Estimation of severity of pinkeye infections were obtained in either August (n = 382) or October (n = 371). 1. DNA was extracted as described in “Current Protocols in Human Genetics” (Dracopoli et al., 2000). 4. DNA was amplified in a 15μL PCR reaction that contained 50 ng genomic DNA, 5.5 μL of distilled water, 7.5 μL of of GoTaQ Master Mix (Promega) and 0.5 μL of both forward and reverse primers. The PCR program consisted of 2 min at 94˚C, followed by 35 cycles of 94˚C, 56˚C and 70˚C for 30 sec. each and a final 5-min extension at 72˚C. 5. Selected samples were sequenced to identify single nucleotide polymorphism (SNP). 6.Restriction Fragments Length Polymorphism assays were designed to genotype each SNP location. The restriction enzyme used for the Exon 2 SNP was Aci I and ApeK I for the exon 3 SNP. 7.Genotype results were statistically analyzed for associations with pinkeye using the GLM procedures of SAS. In addition to pinkeye, we analyzed each genotype with respect to weight, age of dam, and early weaning of the individual. Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2007 Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371 Abstract Introduction Toll-like receptor 4 (TLR4) is a receptor protein. Its main function is to activate immune responses. Because of its function in identifying pathogenic molecules, it has been implicated in resistance to diseases. To investigate its possible role in resistance to IBK, we evaluated the extent to which Single Nucleotide Polymorphism were associated with pinkeye incidence. We PCR amplified the TLR4 gene of 382 American Angus calves in order to genotype two SNP. One in Int1-Ex 2 (-26) position (G/A) and Ex 3 (1678) position (C/T). Pinkeye phenotypic information was available for two time point – August and October. At the August time point, the Ex 2 SNP could account for 2.2% of the phenotypic variation in incidence of pinkeye. A/A animals exhibited more infection than other G/G animal (65% vs. 28% infection rate). While the Ex 3 SNP could account for.09% of the phenotypic variation, there was no statistical difference between genotypes of ex 3. At the October time point, the Ex 2 SNP followed the same pattern as August, it accounted for 3.1% of the phenotypic variation in pinkeye infection. A/A animals exhibited more infection G/G animals. As for Ex 3, it accounted for only.008% of the phenotypic variation, and again, neither of the three genotypes showed any difference at all regarding pinkeye incidence. These data indicate that there is a close relationship between Ex 2 genotype and incidence of pinkeye. Infectious Bovine Keratoconjunctivitis or pinkeye is an inflammatory eye disease caused by Moraxella bovis. Some of the effects in cattle are excessive tearing, and inflammation of the conjunctiva. There exist many treatments for this disease, but none of them have a genetic approach. Toll-like receptor 4 has been identified as a protein that belongs to a group of receptors called Pattern Recognition Receptors. Its function is to recognize lipopolysaccharides and lipoteichoic acid, serving as one of the first immune response of animals. Because of its function in the immune system, TLR4 can be considered as a candidate gene for resistance to a large number of diseases (White et al. 2003). To determine TLR4 as a candidate, Single Nucleotide Polymorphisms were screened on the TLR4 gene. SNP will be used as markers for the identification of a specific genotype on an individual showing any type of resistance to this disease. Associations of Single Nucleotide Polymorphisms in bovine Toll-like receptor 4 with incidence of Infectious Bovine Keratoconjunctivitis in American Angus calves Manuel A. Ortega 1, Ranjit S. Kataria 2, Dinesh Kumar 2, Richard G. Tait Jr 3., and James M. Reecy 3 1 Industrial Biotechnology, University of Puerto Rico- Mayaguez, 2 National Bureau of Animal Genetic Resources, Karnal, India-132 001 3 Department of Animal Science, Iowa Sate University Dracopoli, N.C., Haines, J.L., Korf, B.R., Morton, C.C., Seidman, C.C., Seidman J.G., Smith, D.R., and Boyle A.L. 2000. Current Protocols in Human Genetics, 3, John Wiley & Sons Inc, New York, pp. A.3B.3, A.3C.2 White S.N., Taylor K.H., Abbey C.A., Gill C.A., Womack J.E. 2003. Haplotype variation in bovine Toll-like receptor 4 and computational prediction of a positively selected ligand-binding domain. Proc Natl Acad Sci U S A 100: 10364- 9 Jim Reecy for giving me the opportunity to work in his lab. Dinesh Kumar and Ranjit Kataria for all their mentoring and support. Mary Sue Mayes for all her help in the laboratory. J.R. Tait for all his statistical guidance. Jennifer Young, James Koltes, and Jessica Frerichs for editing purposes. And everyone else who contributed to my experience at Iowa State. Funding provided by USDA-CSREES & National Beef Cattle Evaluation Consortium References Special Thanks to… SNP Association of the different genotypes with incidence of pinkeye Two SNP were found on bovine TLR4 gene, one in Int1-Ex 2 (-26) position and in Ex 3 (1678) position. The genotype of Ex 2 SNP was G/A and C/T for Ex 3 SNP. For August and October measurements, Ex 2 was identified as a significant source on pinkeye incidence. (Pr <.01) According to the Pr values, Ex 3 did not show any relevance on pink eye incidence. Between the three genotypes A/A, G/A and G/G on Ex 2 SNP, A/A showed a higher infection rate (65% for August, 82% for October). Further genotyping is needed to determined in fact that Ex 2 cause an impact in pinkeye incidence. Conclusions Results Table 1. Exon 2 or exon 3 as sole sources associated with August incidence of pinkeye (Data analyzed separately). Table 2. Exon 2 or exon 3 as sole sources associated with October incidence of pinkeye (Data analyzed separately). Figure. 2 Chromatogram of TLR4 Ex 3 sequence. SNP C/T found on Ex3 (1678) position. Source R-Square F value Pr > F Exon 20.030771 5.84 0.0032 Exon 30.000075 0.01 0.9863 Source R-Square F value Pr > F Exon 2 0.021923 4.23 0.0153 Exon 3 0.000861 0.16 0.8501 Figure.1 Chromatogram of TLR4 Ex 2 sequence. SNP G/A found on Int1-Ex 2 (-26) position. SNP Materials and Methods 1.DNA extraction from White Blood Cells 2. DNA quantification 3. Dilution of DNA samples (50 ng/uL) 4. Amplification of TLR4 DNA (PCR Technique) 5. Sequence DNA to identify Single Nucleotide Polymorphism 6. Restriction Enzyme Digestion of PCR products 7. Data Analysis B A B B BB Figure 3. Infection rate percentage of pinkeye on exon 2 genotypes. Figure 4. Infection rate percentage of pinkeye on exon 3 genotypes.